Storage and secretion of smaller products derived biosynthetically from pro-ACTH/endorphin will be studied in four tissues: mouse pituitary tumor cells (AtT-20/l6v), dissociated primary rat anterior and intermediate pituitary cells, rat hypothalamus (arcuate region) both in situ and in culture. Products to be studied include ACTH, alpha-melanotropin, corticotropin-like intermediate lobe peptide, beta-endorphin, beta- and gamma-lipotropin, and 16K fragment. Existence of an ultra-short loop feedback mechanism will be investigated by measuring secretion of immunoactive or pre-labeled pro-ACTH/endorphin-related material with and without added ACTH/endorphin-related peptides. Preferential release of newly-synthesized molecules will be studied with sequential incubations in (3H)-and (35S)methionine, followed by immunoprecipitation. The possible phosphorylation of beta-lipotropin (or other pro-ACTH/endorphin-related peptides) will be determined by incubation in (32P)inorganic phosphate. Coordinate, equimolar secretion of the set of smaller pro-ACTH/endorphin-derived products characteristic of each tissue will be investigated with sequential immunoprecipitations after a prelabeling incubation. Subtle but biologically important alterations in the structure of secreted molecules will be investigated using high pressure liquid chromatography. What appears to be a secretory protein released along with hormone by pituitary tumor cells will be purified and used to raise monoclonal antibodies; secretion of a similar protein by other ACTH/endorphin secreting tissues will be studied. The role of carbohydrate sidechanis in metabolism of pro-ACTH/endorphin will be investigated using lectin-resistant variants of mouse tumor cells. Secretory granules from tumor cells and pituitary tissue will be prepared by subcellular fractionation and their role in the secretory process will be studied. Long-term primary tissue cultures of anterior and intermediate pituitary cells will be studied to determine the effects of denervation, reinnervation, glucocorticoids and corticotropin releasing factor on ACTH/endorphin cells.